Practical Exam Material

Aseptic technique

1. quadrant streaking- loop goes through bunsen burner and then streak 1-2 times and then repeat process 4 times- goal is to perform pure colonies

   • during this make sure to wear proper PPE

   • keep lids OFF the bench

   • minimizing contamination

2. Liquid culture

   1. Transferring pure colony into broth by reflaming loop

   2. also lids NOT on bench top!

   3. CLOSE THE TIP BOX

   4. Chunking- scapel to cut out agor. flame it first

   5. don’t leave plate open

   6. do not put loop down or keep it in air

   7. resterilize loop immediately after

      NEMATODES

      1. keep bunsen burner on at all times while the lids are off the plates

      2. sterilize the worm pick before and after touching each worm

Labeling samples

THREE THINGS GIRL DON’T FORGET

• initials, the date, and the name of the sample

Proper Use of Lab Equipment

Balance: make sure to tare to 0 and then weight and then close

analytical balance: it has a screen on it make sure to close it- for SMALL ass measurements (0.01 mg)

incubator

provides a controlled environment for cell or microbial culture, maintaining specific temperature, humidity, and gas levels

shaking incubator

agitate or shake cell cultures

fridge freezer

used to store samples at low temperatures, capable of freezing items as well as refrigeration.

spectrophotometer

https://upload.wikimedia.org/wikipedia/commons/0/03/Spectrophotometer_Model_1.JPG measures the amount of light that a sample absorbsIt is commonly used to determine the concentration of solutes in a solution.

NEEDS a blank

click the number you wanna measure (1,2,3,4,5)

Nanodrop

know how to read a nanodrop

SHOWS ABSORBANTS at various wavelengths (concentration and measurement of your impurity) 

260/280 ratio of dna to protein

> 1.8 RATIO OF DNA

aim for > 20 ng/mL

microcentrifuge

for the actual tube it needs to be the same amount of liquid

make sure they are 7 apart

across from each other (orientation)

micropipettes

 fair game may be asked to pipette, choosing one, closing the tip box

go down to the first to pick up liquid, second to expel it

serological pipettes

accurately measuring and transferring liquid volumes, typically from less than 1 ml to up to 50 ml

stereoscopic microscope

3D view of a sample, typically used for dissection or examining surface details.

compound microscope

Compound microscopes are used for observing very small, transparent specimens at high magnification (40x-1000x).

inner large one- coerce

outer one - fine

thermocycler

PCR

DNA sequencing, cloning, generation of probes, quantification of DNA and RNA, studying patterns of gene expression, detection of sequence-tagged sites, and many more techniques.

op50

PINKKKKK basillcus

circular

e.coli

NO SPORES

G-

= worm food

4A4-

PURPLE, bacillus

squares

Bt

G+

purple

firms spores

toxic- not worm food they will die

Explain the purpose of quadrant streaking

a technique used to obtain pure cultures on agar medium

Describe the purpose of pure colonies

study the properties of the species/ strain

Compare and contrast a male and hermaphrodite

hermaphrodites are capable of self fertilization and mating with males, they have XX chromosomes. much larger

males can mate with hermaphrodites, X0 gonads, small and have a tail fin

Describe how nematodes are cultured in the lab

they are cutlured in agar rows containing petri dishes on the lawn of the bacteria E. coli

purpose of light microscopy

to visualize and study small structures and samples by creating a magnified image of how they interact with visible light

Gram Staining

• to differentiate between two typs of bacteria (G-) (G+)

• stains the peptidoglycan within the cell wall

  List the components needed for a successful reaction in the correct order used in the protocol, and describe their functions

  • cells are fixed to slide surface

  • crystal violet is added (covers entire slide)

    • this sits and is washed off

    • its function is to stain the cells

  • IODINE- added and covers the whole slide

    • this sits and then is washed off

    • function is to bind stain the G+ cells, helps increase stain retention

  • wash off with ethanol

    • functions as a solvent and will completely wash outdie of thin peptidoglycan

  • add safranin

    • functions in staining the G- cells, staining the bacteria that has been decolorized

GRAM STAINING : Interpret and analyze data obtained through this method (Did you get the expected staining pattern? If not, what might be the reason?

G+ cells= DARK PURPLE

G- cells= pink/ reddish

if no color= a step was skipped or heat wasn’t applied to sample before it was stained

if G+ is pink then= over decolorization with ethanol

if G- is dark purple= under decolorization with ethaol (Wasn’t fully washed)

DNA Purification

purpose: to amplify a specific gene

1. PCR water (volume of the reaction- thing that the reaction takes place in)

2. Template- result of the dna protocol

3. BEF 1 and BER 1 (FORWARD AND REVERSE)- goal to stick to strands

4. dNTPs = ATCG- nucleotides to synthesize new DNA

5. Rx buffer- buffer

6. PFU = polymerase enzyme- MUST BE LAST!!!!

Whole procedure done on ICE ICE ICE!!

GEL ELECTROPHORESIS: Interpret and analyze data obtained through this method (Did you get the expected staining pattern? If not, what might be the reason?

expected product is a single clear band that appears as the expected size compared to DNA ladder

band in negative color= contamination

faint band= low concentration of DNA

smear instead of sharp band= degraded dna, overloaded the sample

gel electrophoresis: purpose

to separate and visualize DNA based on their size and charge

Demonstrate how to correctly set up a gel and gel apparatus

• precast or handmade gel

• samples loaded into wells of gel

  • need to be mixed with loading dye so you can visualize

• cover the gel with TAE buffer

• always run to red

  • DNA is negatively charged

Nematodes

N2 is wild type for c. elegans

XX vs. X0

Go through 4 larval stages

L1 and L2 will go into dauer stage, very skinny, go into status

C. elegans are free living, not parasitic